The overall aim of this project is to characterize the human lysosomal lipases and phopholipases, to examine their genetic deficiencies and to explore their role and pathogenesis of atherosclerosis. Our observation that apolipoprotein C-III activates sphingomyelinase suggests the possibility of physiological roles for extracellular proteins as activators of lysosomal enzymes. Recent studies reported in this revised application demonstrate lysosomal metabolism of phospholipids by intact cultured fibroblasts. To date these studies have demonstrated 1, differences in Niemann-Pick A and B fibroblasts in cell culture, 2, a role for various lipoprotein fractions in determining the uptake and metabolism of phospholipids and 3, abnormalities of HDL mediated uptake of phospholipid by certain familil hypercholesterolemia fibroblasts. We will study the metabolism of radioactive phospholipids in cultured fibroblasts from patients with degenerative diseases. We will examine the effect of various lipoprotein fractions on uptake and metabolism of phospholipids by cultured fibroblasts from patients with degenerative diseases and with familial hypercholesterolemia. We will evaluate the physiological significance of activation of sphingomyelinase by apo C-III using protein modification techniques and using reconstituted lipoproteins to examine the effect on metabolism in cultured fibroblasts. We will search for a lysosomal enzyme which hydrolyzes glycerophosphorylcholine and for enzymes which hydrolyze ether lipids. We will use HPLC to fractionate lysosomal hydrolases, and we will use continuous enzyme monitoring with fluorescent substrates as well as specific assays with radioactive natural substrates to monitor the fractionations. We believe recently developed techniques for studying phospholipid metabolism by cultured fibroblasts promise to yield interesting information regarding lysosomal phospholipases and lipoprotein mediated uptake of phospholipids.